| Background and objective Talaromyces marneffei(TM)infection is one of the most important causes of death among the AIDS population in the south of Yangtze River in China.The risk of death of HIV patients with TM infection in Guangxi is the highest among all opportunistic infections.The reason for the high fatality rate of TM is the existence of a set of immune escape strategies,which is called the"macrophage paradox".Macrophages,as the body’s first line of defense against pathogen invasion,instead provide TM with a niche to avoid immune killing,but its internal mechanism is unknown.Mp1p protein is a virulence factor of TM.Previous studies have shown that Mp1p can affect the host immune response and may play an important role in TM immune escape.However,its molecular mechanism is still unclear and requires further research.In order to solve the above problems,this study aims to explore the role of Mp1p protein in TM immune escape and the potential molecular mechanism.Methods First,the purified Mp1p protein was expressed and purified in vitro and incubated with THP-1 macrophages and Kupffer cells and infected with TM respectively.Colony forming units(CFU)was used to analyze the effect of purified Mp1p protein on the anti-TM ability of macrophages.Lentivirus was used to construct THP-1 macrophage cell line(THP-1-Mp1p~+)and Kupffer cell line(Kupffer-Mp1p~+)stably expressing Mp1p protein.After infection with TM,the CFU method was used to analyze the anti-TM ability of Mp1p on macrophages.Subsequently,the total RNA of THP-1-Mp1p~+cell line and THP-1macrophages infected with TM was extracted for RNA sequencing(RNA-seq).Analyze the differential gene expression profiles shared by macrophages overexpression of Mp1p and TM infection,and use GO,KEGG and GSEA to enrich related pathways to screen out candidate genes and pathways related to Mp1p’s influence on the antibacterial function of macrophages.Finally,the key factors for screening were verified at the gene level and protein level:Western Blot was used to detect the total ERK protein and its phosphorylation level.Real-time fluorescent quantitative PCR(q-PCR)was used to detect the m RNA expression levels of inflammatory factors IL-1β(Il-1β),TNF-α(Tnf-α),and anti-inflammatory factor IL-10(Il-10).Cytokine microsphere detection technology(CBA)and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression levels of IL-1β(Il-1β)and TNF-α(Tnf-α)in the cell supernatant.Results 1.Effect of Mp1p purified protein on the antibacterial ability of macrophages.The CFU of THP-1 macrophages pretreated with Mp1p purified protein and the control group were(2.30±0.16)×10~5CFU and(2.20±0.05)×10~5CFU,respectively.The results were not significantly different(p>0.05).The colony counts of Kupffer cells pretreated with Mp1p purified protein and the control group were(2.60±0.33)×10~5 CFU and(2.30±0.16)×10~5 CFU,respectively.The results had no significant differences(p>0.05).2.Detection of antibacterial ability of Mp1p-overexpressing macrophages.Compared with the negative control group,the CFU of THP-1-Mp1p~+macrophages and Kupffer-Mp1p~+cells infected with TM increased.The CFU after 24h infection were(1.70±0.32)×10~5 CFU,(1.95±0.39)×10~5 CFU,and the CFU after 48h infection were(1.58±0.25)×10~5 CFU,(3.35±0.59)×10~5 CFU,respectively.The differences were statistically significant(p<0.05).3.Analysis of differential gene expression in THP-1-Mp1p~+macrophages.Compared with the negative control group,the expression of inflammatory factors IL-1βand TNF-αin THP-1-Mp1p~+macrophages was significantly reduced,and the expression of MAPK3(ERK1),a key factor of the MAPK pathway,was significantly reduced.The differences were statistically significant(p<0.05).The enrichment of GO,KEGG,and GSEA showed that differential genes were enriched in TNF signaling pathway,IL-17 signaling pathway and MAPK signaling pathway.All three pathways were significantly inhibited(p<0.05).4.Analysis of differential gene expression in THP-1 macrophages infected with TM.Compared with uninfected cells,the expression of IL-6 and IL17 family inflammatory factors in THP-1 macrophages infected with TM was significantly reduced(p<0.05)and were enriched in IL-17 signaling pathway.GSEA analysis showed that this signal pathway was significantly inhibited(p<0.05).5.Overexpression of Mp1p inhibits ERK activation and inflammation in macrophages.The phosphorylation level of ERK protein in THP-1-Mp1p~+macrophages and Kupffer-Mp1p~+cells decreased by 50%and 40%,respectively(p<0.05).The m RNA expression level of IL-1βdecreased by 40%and 30%,respectively(p<0.05).The m RNA expression level of TNF-αdecreased by 50%and 30%,respectively(p<0.05).The m RNA expression level of IL-10 increased9-fold and 2-fold,respectively(p<0.05).The secretion level of IL-1βin the cell supernatant decreased by 83%and 32%,respectively(p<0.05).The secretion level of TNF-αin the supernatant of the two cells decreased by 35%.The difference was statistically significant(p<0.05).Conclusion TM infection may reduce the phosphorylation level of macrophage ERK protein through its virulence factor Mp1p protein and promote the expression of macrophage anti-inflammatory factors and inhibit the secretion of inflammatory factors,thereby evading immune killing.The research uses Mp1p as the entry point to explain the molecular mechanism of TM immune escape from the perspective of macrophage inflammatory response,which can provide new intervention targets and treatment strategies for optimizing the treatment of HIV/TM patients. |
